Pharmacological Characterization of [ 3 H]-Ifenprodil Binding to Polyamine Binding Sites on Rabbit and Rat Retinal Homogenates: Role in Neuroprotection?

Abstract

ABSTRACT

Polyamine binding sites (PBS) represent one of the modulatory sites on the N–methyl–D–aspartate (NMDA) receptor–channel complex. We have characterized [³H]–ifenprodil binding to the PBS on washed homogenates of rabbit and rat retinas. Specific binding of [³H]–ifenprodil (2 nM) (in the presence of 3 μM 1,3–Di [2–tolyl] guanidine HCl and 10 μM GBR12909 to block sigma sites) comprised 47–56% of the total binding. Scatchard analyses indicated interaction with apparent high– and low– affinity sites: dissociation constants (K_ds) = 0.5–0.6 μM and apparent density of sites (B_max) = 1.5–4.3 pmol/mg protein and K_ds = 2.0–2.9 μM, and B_max values = 15.8–17.8 pmol/mg protein (n = 3). Ifenprodil (K_i = 0.4–0.8 μM), eliprodil (K_i = 0.7–0.8 μM), spermine (K_i = 72–79 μM), spermidine (K_i = 283–330 μM), putrescine (K_i > 650 μM) and MK–801 (K_i > 1 mM) (n = 3–5) differentially competed for [³H]–ifenprodil binding. The biphasic competition curves for ifenprodil were resolved into two binding components: rat retinas, IC_50high = 0.19 ± 0.13 μM and IC_501ow = 8.7 ± 1.3 μM; rabbit retinas, IC_50high = 0.1 ± 0.01 μM and IC_50low = 16.0 ± 7.8 μM. These studies have shown the presence of specific PBS labeled by [³H]–ifenprodil in the rabbit and rat retinas which may, in part, be responsible for mediating the neuroprotective effects of eliprodil and ifenprodil.